硝化细菌群的富集及其去除城镇寡营养河道中氨氮污染的应用

Enrichment of nitrifying bacteria and their application in removing ammonia nitrogen pollution in an urban oligotrophic river

摘要:

[背景] 随着工农业的发展，污水排放导致的氨氮超标逐渐成为水体污染的重要因素，脱氮已成为人们研究的重点。目前脱氮方法主要集中于硝化细菌的硝化作用，其将氨氮转化为硝酸盐氮，从而减少水体中氨氮的污染。由于工业废水和农业污水中的有机物含量较高，而且异养硝化细菌具有生长较快等优势，因此对异养菌的研究多于自养菌。然而现有的异养硝化细菌对有机物含量低的自然河道氨氮的去除率不够理想，尤其是富集来自寡营养河道的菌群对其原位环境中氨氮的去除研究尚未见报道。[目的] 富集自然河道中具有硝化功能的菌群并验证其生长情况，检测其菌群结构。研究菌群在人工模拟污水中对氨氮的去除情况，并研究其在自然河道中对氨氮的去除情况。[方法] 从自然河道中使用氨氧化细菌和亚硝酸盐氧化细菌的无机盐培养基进行氨氧化菌群和亚硝酸盐氧化菌群的富集，对富集得到的菌群进行16S rRNA基因测序并分析其菌群结构。采用显微计数法检测菌群的生长情况。模拟自然环境，在桶内置入人工污水，并投入氨氧化菌群AOB1和亚硝酸盐氧化菌群NOB1，于室温下曝气，检测2种菌群共同作用对氨氮的去除情况。将2种菌群同时投入河道水中，通过对河道水取样，观察2种菌群共同作用下水质及氨氮的变化情况。[结果] 富集得到氨氧化菌群AOB1和亚硝酸盐氧化菌群NOB1，获得2个菌群的菌群结构：在氨氧化菌群AOB1和亚硝酸盐氧化菌群NOB1中均为变形菌门（Proteobacteria）的细菌相对丰度最高，分别占99.28%和99.64%；在菌群AOB1中相对丰度较高的属是Aeromonas （73.00%）和Delftia （9.17%），在菌群NOB1中相对丰度较高的属是Aeromonas （36.66%）和Pseudomonas （30.82%）。两个菌群均生长良好，以5%接种量进行转接时，菌群AOB1和NOB1分别于2-3 d生物量达到最大（分别为5.23×10¹⁰个/L和3.63×10¹⁰个/L）。菌群处理寡营养（葡萄糖浓度0.04 g/L）的人工生活污水1周后对氨氮的去除率为95.26%，而且无亚硝酸盐氮的积累。取自然河道水于桶内，向其中投放菌群AOB1和NOB1，于实验室内曝气（曝气量5 L/min），10 d后氨氮的去除率为94.04%，无亚硝酸盐氮的积累。将菌群投入浙江省台州市某河道700 m区域内，在有少量排污的情况下，16 d氨氮的去除率为49.19%，氨氮的去除率较好。[结论] 本研究富集了来自寡营养河道中具有硝化功能的2个菌群，其共同作用可以去除实验室模拟污染水体和寡营养河道中的氨氮。本研究储备了微生物资源，证明了利用微生物消除城镇寡营养河道氨氮污染的可行性，对实现国家“美丽乡村”的建设目标具有潜在的示范价值。

Abstract:

[Background] With the development of industry and agriculture, excessive ammonia nitrogen caused by sewage discharge has gradually become an important issue in water pollution, and ammonia nitrogen removal has become one of research hotspots. In previous studies, it has been reported that nitrifying bacteria were able to convert ammonia nitrogen to nitrate nitrogen, resulting in the reduction of ammonia nitrogen concentration in water. Thus the ammonia nitrogen elimination in polluted water by biological methods mainly depended on nitrifying bacteria. Currently there are more studies on heterotrophic nitrifying bacteria than autotrophic ones. However, the removal efficiency of the existing heterotrophic nitrifying bacteria for ammonia nitrogen in oligotrophic rivers is not ideal. In particular, the bioremediation in situ by nitrifying consortia from oligotrophic rivers has virtually not been reported. [Objective] We aim to enrich and characterize nitrifying consortia from oligotrophic rivers, and explore the potential of the ammonia nitrogen elimination in an oligotrophic river by in situ isolated consortia. [Methods] The mineral media for ammonia-oxidizing bacteria and nitrite-oxidizing bacteria are used to isolate ammonia-oxidizing and nitrite-oxidizing bacterial consortia from rivers. The microbial diversities of two consortia were identified according to sequences of V3-V4 region of 16S rRNA genes. The microscopic counting method was used to detect the growth of the consortia. Two consortia were inoculated into the artificial sewage together with aerating at room temperature, and the depletion of ammonia nitrogen was detected. Two consortia were together introduced into the oligotrophic river in situ, and the depletion of ammonia nitrogen was also detected. [Results] In this experiment, an ammonia-oxidizing bacterial consortium and a nitrite-oxidizing bacterial consortium were enriched and designated AOB1 and NOB1, respectively. In AOB1 and NOB1, 99.28% and 99.64% strains belong to Proteobacteria, respectively. Furthermore, the relatively dominant genera in AOB1 are Aeromonas (73.00%) and Delftia (9.17%). The relatively dominant species in NOB1 are Aeromonas (36.66%) and Pseudomonas (30.82%). After 2-3 d incubation in the mineral media, the biomasses of AOB1 and NOB1 reached to 5.23×10¹⁰ cells/L and 3.63×10¹⁰ cells/L, respectively. When AOB1 and NOB1 were introduced into artificial sewage with low organic matter (glucose concentration being 0.04 g/L), the depletion of ammonia nitrogen was 95.26% after 7 d, without accumulation of nitrite nitrogen. When AOB1 and NOB1 were introduced into water from an oligotrophic river (Taizhou city, Zhejiang province) with aerating (areation rate being 5 L/min) in the lab, and the depletion of ammonia nitrogen was 94.04% after 10 d. AOB1 and NOB1 were also together introduced into a 700-meter water area of the above river, and sewage was still discharged continuously into this river during the testing period. After 16 d, 49.19% ammonia nitrogen in the above water area were removed.[Conclusion] This study has enriched an ammonia-oxidizing bacterial consortium and a nitrite-oxidizing bacterial consortium from oligotrophic rivers, which can together remove ammonia nitrogen in artificial sewage and oligotrophic river water. This study provides microbial resources and a feasibility of using microbes to eliminate ammonia nitrogen pollution in urban oligotrophic rivers. It also has a potential application in implementing the desired goal of "The Beautiful Countryside".